Clinical analysis on steroids. XII. Occurrence of D-homoannulation during the hot acid hydrolysis of 5β-pregnane-3α,20α-diol disulfate

Two D-homosteroids were isolated from the hydrolyzate of 5β-pregnane -3α,20α-diol disulfate (II) when it was refluxed in 3N hydrochloric acid. The structures of these steroids have been elucidated as 17α-methyl-D-homo-5β-androstane-3α, 17aβ-diol (VI) and 17α-methyl-17aγb-chloro-D-homo-5β-androstan-3α-ol (VIII) by instrumental analyses. The former was identical with a synthetic specimen derived from 5β-pregnane-3α,20β-diol di-sulfate (IV) by uranediol rearrangement. The main hydrolyzates obtained were 17α-ethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (V) and 5β-pregnane-3α, 20α-diol (III).     

Radioimmunoassay of ursodeoxycholic acid in serum

A sensitivie and specific radioimmunoassay for the measurement of serum ursodeoxycholic acid has been developed. Ursodeoxycholic acid bound to bovine serum albumin was used as an antigen, and antiserum to this antigen was raised in the rabbit. [11, 12-3h2]Ursodeoxycholic acid was used as the radioactive tracer, and the radioimmunoassay was carried out by the method of Simmonds et al. (1973. Gastroenterology. 65: 705-711). The percentage of bound radioactivity decreased linearly with a logarithmic increase in unlabeled ursodeoxycholic acid from 10 to 200 pmol. The antiserum showed extremely high specificity for ursodeoxycholic acid (free and conjugated), and the values determined by radioimmunoassay indicated a close correlation with those found by gas-liquid chromatography. In normal Japanese subjects, a small amount of ursodeoxycholic acid in serum was detected, and the level was detected, and the level was 0.15 +/- 0.11 nmol/ml. This convenient radioimmunoassay will provide useful information about the metabolism of ursodeoxycholic acid in man.     

Activation of phosphodiesterase by rhodopsin and its analogues

Activation of guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase (EC 3.1.4.35.) in frog rod outer segment membrane by rhodopsin and its analogues was investigated. The Schiff-base linkage between opsin and retinal in rhodopsin was not always necessary for the phosphodiesterase activation. The binding of beta-ionone ring of retinal to a hydrophobic region of opsin was not enough to induce the enzyme activation. A striking photo-activation of the enzyme was induced by photo-isomerization of rhodopsin analogues from cis to trans form. It seems probable that an "expanded" conformation of opsin around the retinylidene chromophore induced by the cis to trans isomerization may be the trigger for the activation of phosphodiesterase. On the other hand, the phosphodiesterase in frog rod outer segment was activated by warming of bathorhodopsin to -12 degrees C and then incubating it at the same temperature. Thus, metarhodopsin II or an earlier intermediate than metarhodopsin II should be a direct intermediate for the enzyme activation.     

Arachidonate metabolism in bovine gallbladder muscle

Incubation of [1-¹⁴C]arachidonic acid (AA) with homogenates of bovine gallbladder muscle generated a large amount of radioactive material having the chromatographic mobility of 6-keto-PGF_1α (stable product of PGI₂) and smaller amounts of products that comigrated with PGF_2α and PGE₂. Formation of these products was inhibited by the cyclooxygenase inhibitor indomethacin. The major radioactive product identified by thin-layer chromatographic mobility and by gas chromatography - mass spectrometric analysis was found to be 6-keto-PGF_1α. The quantitative metabolic pattern of [1-¹⁴C]PGH₂ was virtually identical to that of [1-¹⁴C]AA. Incubation of arachidonic acid with slices of bovine gallbladder muscle released labile anti-aggregatory material in the medium, which was inhibited by aspirin or 15-hydroperoxy-AA.These results indicate that bovine gallbladder muscle has a considerable enzymatic capacity to produce PGI₂ from arachidonic acid.     

Comparison of cytochrome P-450 species which catalyze the hydroxylations of the aromatic ring of estradiol and estradiol 17-sulfate

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E₂) and estradiol 17-sulfate (E₂-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E₂. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E₂ was converted to such metabolites as estrone and those having a hydroxyl group at positions 6β, 15 or 16, each production of which was estimated to be catalyzed by single or multiple P-450s.     

Vimentin is hyperphosphorylated in primary human fibroblasts treated with okadaic acid.

Okadaic acid and dinophysistoxin-1 (35-methylokadaic acid) induced hyperphosphorylation of a 58 kDa protein in primary human fibroblasts, due to inhibition of protein phosphatase 1 and 2A activities. The protein was present in the nuclear and cytosolic fractions. Its pI was 5.3. The hyperphosphorylated protein reacted with monoclonal and polyclonal anti-vimentin antibodies, but not with anti-nucleolin antibody. Phosphorylation of vimentin was stimulated in vitro by dinophysistoxin-1 dose-dependently in the presence of protein phosphatase 2A and protein kinases.     

Differences in the photobleaching process between 7-cis- and 11-cis-rhodopsins: a unique interaction change between the chromophore and the protein during the lumi-meta I transition.

The photochemical and subsequent thermal reactions of 7-cis-rhodopsin prepared from cattle opsin and 7-cis-retinal were investigated by low-temperature spectrophotometry and laser photolysis, and compared with those of 11-cis-rhodopsin prepared from cattle opsin and 11-cis-retinal. Low-temperature experiments revealed that the absorption maxima of batho and lumi intermediates from 7-cis-rhodopsin were at slightly shorter wavelengths than those of 11-cis-rhodopsin while the meta I intermediates of both rhodopsin isomers showed the same absorption maxima. Kinetic experiments of the photobleaching process of 7-cis-rhodopsin using picosecond and nanosecond laser pulses revealed the formation of intermediates corresponding to the batho, lumi, meta I, and meta II intermediates from 11-cis-rhodopsin. An intermediate of 7-cis-rhodopsin corresponding to photorhodopsin (a precursor of bathorhodopsin), however, was not detected. Batho and lumi intermediates from 7-cis-rhodopsin had shorter lifetimes (approximately 40 ns and 300 microseconds) than those of 11-cis-rhodopsin (250 ns and 800 microseconds), but the lifetime of the meta I intermediate from 7-cis-rhodopsin was identical with that from 11-cis-rhodopsin (12 ms). These results indicate that the difference in configuration of the original chromophore between 7-cis- and 11-cis-rhodopsins is a cause of different chromophore-opsin interactions in the batho and lumi stages, while in the meta I stage the difference has disappeared by the relaxation of the protein near the chromophores. A possible interaction change between the 9-methyl group of the chromophore and its neighboring protein during the lumi-meta I transition will be discussed.     

Phosphorylation of iodopsin, chicken red-sensitive cone visual pigment

The amino acid sequence has been determined for the carboxyl-terminal 41 amino acids of chicken red-sensitive cone pigment, iodopsin. This sequence is distinct from but structurally homologous to that of other visual pigments. It contains a region rich in the hydroxy amino acids serine and threonine. In the related rod cell visual pigment, rhodopsin, such serines and threonines have previously been identified as sites for phosphorylation by rhodopsin kinase. Phosphorylation of photolyzed rhodopsin serves to terminate its ability to function in visual transduction as an activator of G-protein. We have purified and reconstituted both chicken rhodopsin and chicken iodopsin and shown them to be phosphorylated by bovine rhodopsin kinase. Chicken iodopsin has a Km and Vmax similar to but distinguishably different from that for bovine rhodopsin. These results, in conjunction with other data, suggest that visual pigments in cone cells, upon absorption of light, undergo functional processes similar to those of the visual pigments in rod cells.     

Pulse sequences generated by a degenerate analog neuron model

The response characteristics of an electronic neuron model proposed by the authors are investigated. Periodic stimulating pulse sequences with a fixed frequency are applied to the analog neuron model and the response pulse sequences are studied. In the degenerate case, the state transition of the neuron model during one period of the stimulating pulse sequence is described by a first order piecewise linear difference equation with a jump. It is shown that the periodic response pulse sequences of the neuron model belong to a special class of pulse sequences generated by a simple algorithm, and that the relation between the pulse width (or amplitude) of the stimulating pulse and the firing rate of the neuron model takes the form of an extended Cantor function.     

Circular dichroism of squid rhodopsin and its intermediates

Circular dichroism (CD) and absorption spectra of squid (Todarodes pacificus) rhodopsin, isorhodopsin and the intermediates were measured at low temperatures. Squid rhodopsin has positive CD bands at wavelengths corresponding the - and β-absorption bands at liquid nitrogen temperature (CD maxima: 485 nm at -band and 348 nm at β-band) as well as at room temperature (CD maxima: 474 nm at -band and 347 nm at β-band). The rotational strength of the -band has a molecular ellipticity about twice that of cattle rhodopsin. The CD spectrum of bathorhodopsin displays a negative peak at 532 nm, the rotational strength of which has an absolute value slightly larger than that of rhodopsin. The reversal in sign at -band of the CD spectrum may indicate that the isomerization of retinal chromophore from twisted 11-cis form to twisted 11-trans form has occurred in the process of conversion from rhodopsin to bathorhodopsin. Lumirhodopsin has a small negative CD band at 490 nm, the maximum of which lies at 25 nm shorter wavelengths than the absorption maximum (515 nm), and a large positive CD band near 290 nm, which is not observed in rhodopsin and the other intermediates. This band may be derived from a conformational change of the opsin. In the process of changing from lumirhodopsin to LM-rhodopsin, the CD bands at visible and near ultraviolet regions disappear. Both alkaline and acid metarhodopsins have no CD bands at visible and near ultraviolet regions.